N-acetylmannosamine dehydrogenase, process for its production, method for quantitatively analyzing N-acetylmannosamine or sialic acid, and kit for the quantitative analysis

ABSTRACT

N-Acetylmannosamine dehydrogenase which takes off hydrogen from N-acetylmannosamine to convert it into N-acetylmannosaminolactone and, at the same time, reduces coenzyme NAD into NADH. This enzyme can be obtained by culturing, in a medium, a strain belonging to Genus Flavobacterium and having an ability to produce N-acetylmannosamine dehydrogenase producing activity, and then collecting it. This enzyme is usable in the quantitative analysis of N-acetylmannosamine or sialic acid.

This application is a division of application Ser. No. 121,916, filedNov. 17, 1987, now U.S. Pat. No. 4,960,701.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to novel N-acetylmannosamine dehydrogenase(hereinafter referred to as N-AMDH) which acts upon N-acetylmannosamine(hereinafter referred to as N-AM) to convert it intoN-acetylmannosaminolactone and, at the same time, reduces nicotinamideadenine dinucleotide (NAD) into reduced nicotinamide adeninedinucleotide (NADH), as well as to a process for producing N-AMDH, anenzymatic method for quantitatively analyzing N-AM or sialic acid(hereinafter referred to as SA), and a kit for the quantitativeanalysis. 2. Description of the Prior Art

In the current clinical tests, SA in serum is measured, and this playsan important role in the diagnoses of acute and chronic inflammations,shocks, trauma, myocardial infarction, diabetes mellitus, liverdiseases, cancers, etc.

The measurements of SA are roughly classified into chemical method andenzymatic method.

The chemical method is being gradually replaced by the enzymatic methodof higher accuracy, because chemical method is inferior in variouspoints such as specificity, workability, dangerousness of the agentsused in its, etc.

Until today, method A and method B have been proposed as the enzymaticmethod, according to a rough classification.

Methods A and B are identical in that neuraminic acid aldolase isreacted upon SA to decompose the latter into N-AM and pyruvic acid.However, they are different from each other in that according to methodA, N-AM is treated with acylglucosamine-2-epimerase andN-acetylhexosamine oxidase to form hydrogen peroxide and the latter isanalyzed, while according to method B, pyruvic acid is treated withpyruvic acid oxidase or lactic acid dehydrogenase (LDH) to form hydrogenperoxide or NADH, respectively, and they are analyzed.

Method A is disadvantageous in that the existence ofacylglucosamine-2-epimerase complicates the system, and method B isdisadvantageous in that it is influenced by the endogenous pyruvic acid.

SUMMARY OF THE INVENTION

The present inventors studied a measurement of SA which is easy tooperate and has a high accuracy. As the result, it was found that abacterial strain belonging to Genus Flavobacterium isolated from thesoil produces a novel enzyme which, when reacted with N-AM, converts thelatter into N-acetylmannosaminolactone and, at the same time, reducesNAD into NADH, and this enzyme is effectively utilizable in themeasurement of SA. Based on this finding, the present invention wasaccomplished.

Thus, the present invention provides a novel enzyme N-AMDH which, whenreacted with N-AM, converts the latter into N-acetylmannosaminolactoneand, at the same time, reduces NAD into NADH. Further, the inventionalso provides a process for producing N-AMDH which comprises culturing astrain belonging to Genus Flavobacterium and having an ability toproduce N-AMDH in a medium and collecting N-AMDH from the culturedproduct. Further, the invention also provides a method forquantitatively analyzing N-AM which comprises treating anN-AM-containing sample with N-AMDH and determining the resulting NADH.Further, the invention also provides a method for quantitativelyanalyzing SA which comprises treating an SA-containing samplesuccessively or simultaneously with N-acetylneuraminic acid aldolase andN-AMDH and determining the resulting NADH. Further, the invention alsoprovides a quantitative analysis kit comprising at least N-AMDH, NAD anda buffer solution.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, FIG. 1 is a graph illustrating the optimumpH value of this enzyme; FIG. 2 is a graph illustrating its stable pHrange; FIG. 3 is a graph illustrating the temperature range suitable forthe action of this enzyme; FIG. 4 is a graph illustrating the heatstability of this enzyme; FIG. 5 is a diagram illustrating theelectrophoretic band; FIG. 6 is calibration curve in Example 3; and FIG.7 is calibration curve in Example 7; provided that the buffer solutionsused in FIG. 1 and FIG. 2 were potassium phosphate buffer (-),trishydrochloric acid buffer (Δ-Δ) and glycine-sodium hydroxide buffer( - ).

DETAILED DESCRIPTION OF THE INVENTION

Next, the present invention will be explained below more concretely.

Physico-chemical properties of the novel enzyme N-AMDH used in thepresent invention are as follows.

(1) Action and substrate-specificity

As shown in the following reaction scheme, N-AMDH oxidizes N-AM intoN-acetylmannoaminolactone and, at the same time, reduces NAD into NADHin the presence of N-AM and NAD. ##STR1##

In the presence of water, N-acetylmannosaminolactone is spontaneouslyhydrolyzed to form N-acetylmannosaminic acid. Accordingly, the reactionis substantially irreversible. N-AMDH hardly acts or does not act at allupon other neutral sugars, hexosamine, N-acetylglucosamine andN-acetylgalactosamine, except that is acts upon N-glycolylmannosamine inthe same manner as above. It hardly utilizes nicotinamide adeninedinucleotide phosphate (NADP), 2,6-dichlorophenol indophenol and thelike as electron acceptor.

(2) Optimum pH and stable pH range

Its optimum pH is 8.0 to 9.0, when trishydrochloric acid buffer is used.

FIG. 1 illustrates the results of enzymatic activity measurement usingpotassium phosphate buffer, tris-hydrochloric acid buffer andglucine-sodium hydroxide buffer.

As shown in FIG. 2, its stable pH range is 8.5 to 9.5.

The buffer solutions used there are potassium phosphate buffer,tris-hydrochloric acid buffer and glycine-sodium hydroxide buffer.

(3) Temperature range suitable for its action As shown in FIG. 3, it is35° C. to 50° C.

(4) Conditions (pH, temperature) of inactivation

As shown in FIG. 4, it keeps activity at stable up to a temperature of45° C., when heat treated for minutes. At temperatures exceeding 45° C.,it rapidly loses its activity. When heat treated at 45° C. for 10minutes, it is stable at a pH of 8.5-9.5, while it is particularlyinstable at a pH value of 7 or below.

(5) Influence of inhibitor and stabilization

    ______________________________________                                        Inhibitor      Residual activity (%)                                          ______________________________________                                        None           100                                                            HgCl.sub.2      6                                                             NiSO.sub.4     69                                                             ZnSO.sub.4     71                                                             CuSO.sub.4     79                                                             NaN.sub.3      83                                                             SDS            18                                                             KCN            101                                                            EDTA           89                                                             BSA            96                                                             PCMB           95                                                             Iodoacetamide  87                                                             8-Hydroxyquinoline                                                                           95                                                             o-Phenanthroline                                                                             87                                                             α,α'-Dipyridyl                                                                   97                                                             5'-AMP         76                                                             ______________________________________                                    

The table presented above illustrates the enzymatic activity of N-AMDHmeasured in solutions containing various metallic ions and inhibitors ata concentration of 2 mM. There is known no substance making a particularcontribution to its activation and stabilization.

(6) Method of purification

This enzyme can be isolated and purified according to usual purifyingmeans such as column chromatography using DEAE-cellulose, precipitationusing ammonium sulfate, column chromatography using DEAE-Sephadex,column chromatography using 5'-AMP-Sepharose, gel filtration usingSephadex, and the like, or combinations thereof.

(7) Molecular weight

As measured by gel filtration method using 0.05M tris-hydrochloric acidbuffer (containing 0.1M NaCl) and Sephadex G-200 column, its molecularweight is about 110,000 to 120,000.

(8) Electrophoresis using polyacrylamide gel

As shown in FIG. 5, acrylamide disk electrophoresis using 7.5%polyacrylamide gel gives a nearly single band. The migration distanceafter 80 minutes at 4 mA is 28 mm.

(9) Isoelectric point

As measured by acrylamide gel isoelectric focusing, its isoelectricpoint is 4.9.

(10) Activity measurement

To 1.8 ml of 0.05M tris-hydrochloric acid buffer (pH 8.2) is added 0.1ml of 60 mM NAD solution. After keeping the mixture at 37° C. for 10minutes, 10 microliters of enzyme solution is added, and then 0.1 ml of0.3M N-AM is added. By homogenizing the mixture, the reaction isstarted. The reaction mixture is immediately transferred into a lightabsorbance measurement cell (light path 1 cm) kept at 37° C., andabsorbance is measured over a period of 5 minutes (if necessary, over alonger period of time) at intervals of one minute at a wavelength of 340nm. A quantity of enzyme capable of forming 1 micromole of NADH in oneminute is taken as one unit.

As above, the enzyme of this invention is a novel enzyme which isentirely hitherto unknown in action and substrate-specificity.

Next, the production process of the novel N-AMDH according to thepresent invention will be explained.

The microbial strain used belongs to Genus Flavobacterium and has anN-AMDH-producing ability. One concrete example of such strain isFlavobacterium sp. No. 141-8; and varieties and mutant strains thereofare also usable. Flavobacterium sp. No. 141-8 is a strain which has beenisolated by the present inventors firstly from the soil, and itsbacteriological properties are as follows.

(a) Morphology

Microscopic observation (cultured in sugar-bouillon medium at 30° C. for16 hours)

(1) Size of cell: Rod having a size of 0.45-0.5×0.5-11 microns.

(2) Polymorphism of the cell: The shape ranges from a nearly sphericalform to longer rod-like form, with contamination by short chain-likeconjunction at terminals.

(3) Motility: Non-motile.

(4) Spore: None.

(5) Gram-stain: Negative.

(6) Acid-fast: Negative.

(b) State of Growth in Various Media

(1) Bouillon-agar plate culture (2 days at 30° C.)

Circular colonies are smooth and translucent; 0.5 mm diameter;relatively bad growth.

(2) Sugar-bouillon-agar plate culture (2 days at 30° C.)

Circular colonies are smooth and translucent; 0.8 mm in diameter; milkywhite mucous colonies are formed in 5 days; no pigment produced.

(3) Sugar-bouillon-agar slant culture (2 days at 30° C.)

Milky turbid mucous liquid; in 3 days, the liquid flows down and gathersat bottom.

(4) Sugar-bouillon liquid culture (2 days at 30° C.)

A slight growth

(5) Bouillon-gelatin stab culture (3 days at 30° C.)

A slight growth without liquefaction.

(6) Litmus milk: No change, nor coagulation.

(c) Physiological Properties

(1) Reduction of nitrates: Positive.

(2) Denitrification: Negative.

(3) MR test: Negative, provided that positive in aerobic culture.

(4) VP test: Negative.

(5) Formation of indole: Negative.

(6) Formation of hydrogen sulfide: Negative.

(7) Hydrolysis of starch: Negative.

(8) Utilization of citric acid: Negative.

(9) Utilization of inorganic nitrogen source: Ammonia is utilized, whilenitric acid is not utilized.

(10) Formation of pigment: Negative.

(11) Urease: Positive.

(12) Oxidase: Positive.

(13) Catalase: Positive.

(14) Growing condition range: 15° C.-41° C. (optimum temperature 30°C.), pH 4.5-8.5 (optimum pH ca. 6.5)

(15) Behavior to oxygen: aerobic; a slight growth under anaerobiccondition, too.

(16) O-F test: No change, or a very weak fermentation.

(17) Formation of acid and gas from sugars: * means aerobic culture:

    ______________________________________                                                   *Formation of acid                                                                        Formation of gas                                       ______________________________________                                        (i)  L-Arabinose +             -                                              (ii) D-Xylose    +             -                                              (iii)                                                                              D-Glucose   +             -                                              (iv) D-Mannose   +             -                                              (v)  D-Fructose  +             -                                              (vi) D-Galactose +             -                                              (vii)                                                                              Maltose     +             -                                              (viii)                                                                             Sucrose     +             -                                              (ix) Lactose     +             -                                              (x)  Trehalose   +             -                                              (xi) D-Sorbit    +             -                                              (xii)                                                                              D-Mannit    +             -                                              (xiii)                                                                             Inosit      +             -                                              (xiv)                                                                              Glycerin    +             -                                              (xv) Starch      -             -                                              ______________________________________                                    

(d) Other Characteristics

(1) Penicillin resistance: Growth even at 100 units/ml.

(2) Sodium chloride resistance: No growth above 2%.

(3) Motility of colony rim: No fluidity observed.

(4) Decomposition of Tween 80: Negative.

(5) Decomposition of esculin: Negative.

By comparing the above-mentioned characteristic properties of theabove-mentioned novel N-AMDH-producing strain with the classificationmentioned in "Bergey's Mannual of Systematic Bacteriology (1984) Vol.1", it is considered that this strain belongs to Genus Flavobacteriumbecause it is a gram-negative, aerobic, non-sporeforming bacillus havingno motility, and it is catalase-positive and oxidase-positive and formsacid from many sugars under aerobic conditions, and it is resistant toPenicillin.

Since it forms acid from sugars under aerobic conditions, it isconsidered analogous to Flavobacterium spiritivorum. However, it isdifferent from the latter in the decomposition of esculin, reduction ofnitrate and decomposition of Tween 80. Thus, it can be regarded as anovel strain unknown so far.

For the reasons mentioned above, this strain has been namedFlavobacterium sp. No. 141-8. Flavobacterium sp. No. 141-8 has beendeposited in Fermentation Research Institute, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry,Japan, as FERM BP-1222 under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure.

The medium used in the present invention may be any of synthetic andnatural media, so far as it appropriately contains carbon source,nitrogen source, inorganics and other nutrients. As the carbon source,glucose, galactose, fructose, xylose, glycerin and the like can be used.As the nitrogen source, not only ammonium salts but alsonitrogen-containing organic compounds such as peptone, digested casein,sodium glutaminate, yeast extract and the like are successfully usable.As the inorganics, salts of sodium, potassium, magnesium, manganese,calcium, iron and the like can be used.

In the present invention, N-AMDH is obtained in a high yield when astrain having N-AMDH-producing ability is cultured or dipped in a mediumcontaining N-AM or N-acetylglucosamine. As a preferable example of saidmedium, a medium comprising 0.5% of N-AM, 0.1% of meat extract, 0.5% ofpolypeptone, 0.2% of yeast extract, 0.14% of sodium chloride and 0.1% ofmonopotassium hydrogen phosphate and having a pH value of 6.8 can bereferred to. When the strain is subjected to aeration-agitation culturein this medium at 30° C. for 36 hours, the production titer is 10 to 100times as high as that obtained in a culture using other carbon source inplace of N-AM.

Temperature of the culture is usually 20° to 40° C. and preferably 30°to 33° C. Starting pH of the culture is usually 6 to 8 and preferablyabout 7. Under such conditions, a shaking culture or a submergedagitation culture is carried out for 20 to 40 hours. Otherwise, thebacterial cells which have been grown in other medium suitable for itsgrowth and not containing A-AM or N-acetylglucosamine are dispersed inthe above-mentioned medium at a high concentration aerobically for 1-10hours in the presence of N-AM and N-acetylglucosamine. Thus, N-AMDH isaccumulated in the cultured product or cell suspension.

Since N-AMDH is usually present in bacterial cells, it is preferable toseparate bacterial cells by centrifugation or filtration. By destructingthe cells in an appropriate amount of buffer, the enzyme is solubilizedand released into the solution.

As the means for destructing bacterial cells, physical means such asDynomill, French press, ultrasonic wave and the like, chemical meanssuch as Triton X-100, sodium lauryl sulfate, EDTA and the like, orenzymatic means such as lysozyme and the like is used either alone or incombination. After destructing bacterial cells, nucleic acid is removedin the usual manner and insoluble matter is removed by filtration orcentrifugation, whereby N-AMDH is obtained.

If desired, N-AMDH thus obtained is further purified according to theconventional means for isolation and purification of enzymes such as (1)column chromatography using DEAE-cellulose column, (2) fractionatingprecipitation using ammonium sulfate, (3) column chromatography usingDEAE-Sephadex column, (4) column chromatography using 5'-AMP-Sepharosecolumn, (5) gel filtration using Sephadex, and the like, or theirappropriate combination, whereby a purified N-AMDH can be obtained.

Next, the method for quantitatively analyzing N-AM or SA and thequantative analysis kit of the present invention will be concretelyillustrated.

The principle of the measurement of the invention is as shown below:##STR2##

That is, N-AMDH is reacted with N-AM in a sample, and the formed NADH ismeasured according to well known method of measurement, such asmeasurement of absorbance at 340 nm (ultraviolet region).

Otherwise, it is also possible to measure the formed NADH by contactingsample with various enzymes fixed on a solid. If necessary forpreventing the influence of coexisting LDH, an inhibitor such as oxamicacid, oxalic acid or the like may be added in an appropriate amount.

The N-AMDH used in the invention may be of any origin. Preferably, theN-AMDH obtained by culturing a microorganism, particularly a strainselected from the bacteria belonging to Genus Flavobacterium, is used.

As the above-mentioned enzyme-producing strain belonging to GenusFlavobacterium, Flavobacterium sp. No. 141-8 (FERM BP-1222) can bereferred to, for example.

In reacting N-AMDH with N-AM in a sample, the reaction is carried out ata pH value of 7-10 at a temperature of 50° C. or below, preferably at apH value of 8-9.5 at a temperature of 30°-45° C., and usually for aperiod of about 2 to 20 minutes. For regulating pH value, any buffersolution may be used, so far as it can maintain the above-mentioned pHrange and does not disturb the enzymatic reaction. For example,potassium phosphate buffer, tris-hydrochloric acid buffer,glycine-sodium hydroxide buffer, sodium carbonate buffer and the likeare successfully usable for this purpose.

Though the quantitative analysis of NADH formed by the action of N-AMDHmay be carried out by any methods, the most generally used method ismeasurement of absorbance at 340 nm (ultraviolet region). Apart fromabove, there are a few methods which comprise converting NADH into a dyehaving absorption in the visible region and then determining the dye,such as the method which comprises reacting NADH with phenazinemethosulfate and Nitrobluetetrazolium and measuring the absorption ofthe resulting diformazan at 570 nm, and the method which comprisesreacting NADH with NADH-oxidase (J. Biochem. 98, 1433 (1985)), phenazinemethosulfate or an electron transferring substance or metallic ionexhibiting a similar behavior to form hydrogen peroxide, developing acolor therefrom in the presence of peroxidase and various chromogens,and measuring absorbance at appropriate wavelengths. When NADH isderived into hydrogen peroxide, it is also possible to analyze it bydeveloping a luminescence from it in the presence of luminol. It is alsopossible to analyze NADH semi-quantitatively by adding appropriatelyselected plural redox indicators and electron transferring substancesand observing the color tone. All these method of detection may beselected in accordance with their characteristic features.

In quantitatively analyzing SA, an SA-containing sample is reacted withN-acetylneuraminic acid aldolase to decompose SA into N-AM and pyruvicacid, followed by treating the decomposant solution with N-AMDH andmaking measurement in the same manner as above.

In analyzing SA, the SA in a sample must be in the liberated state. WhenSA is combined with protein or glycolipid as in serum, plasma and sometissues, the SA is once liberated by the action of neuraminidase andthen analyzed. Though the neuraminidase used in this case may be of anyorigin, those produced by microorganisms belonging to Genus Clostridium,Genus Arthrobacter, Genus Corynebacterium, Genus Streptococcus, etc. arepreferable.

Next, the kit of the invention for quantative analysis of N-AM or SA iscomposed of N-AMDH or N-AMDH and N-acetylneuraminic acid aldolase, NAD,enzymes and reagents for quantatively analyzing the formed NADH, andbuffering reagent for smoothly advancing the reaction. These reagentsand enzymes are used in the form of liquid, solid or freeze-driedmaterial, and they are dissolved and mixed into buffer solution beforeuse in accordance with requirement to make a measuring reagent.

In determining N-AM, the kit is directly acted upon N-AM-containingsample to form NADH. The NADH is measured either directly or afteraddition of NADH-analyzing reagents.

In determining SA, N-acetylneuraminic acid aldolase is firstly reactedupon sample to form N-AM. Next, N-AMDH is reacted to form NADH. The NADHis determined either directly or after addition of NADH-measuringreagents. The system for the measurement may be any of single reagentsystem, double reagent system and multi reagent system.

When the novel N-AMDH of the present invention is used, N-AM can bequantitatively analyzed with a high accuracy and based on it thequantity of SA can be known. As its result, various diseases can bediagnosed effectively. Further, according to the invention, N-AM can beexactly analyzed quantitatively without influence of coexistingN-acetylhexosamine, which is quite meaningful in the studies of complexsugars. Similarly, the present invention enables to quantitativelyanalyze SA with a high exactness without influence of endogenous pyruvicacid, which is quite meaningful in the diagnosis based on clinical testsof SA.

Next, the present invention will be illustrated with reference to theExamples.

EXAMPLE 1

300 ml Erlenmeyer flask containing 50 ml of a seed culture medium (pH8.0) containing 0.75% of glucose, 0.2% of yeast extract, 0.5% ofpolypeptone, 0.1% of meat extract, 0.14% of sodium chloride and 0.1% ofmonopotassium hydrogen phosphate was inoculated with Flavobacterium sp.No. 141-8 (FERM BP-1222). After a shaking culture at 30° C. for 24hours, the seed culture fluid was transplanted into a jar fermenter(manufactured by Iwashiya Seibutsu Kagaku K. K.) containing 2 liters ofthe same medium as above and subjected to an aeration (2 liters/minute)agitation (400 rpm) culture at 30° C. for 36 hours. The culture fluidwas centrifuged at 8,000 rpm for 20 minutes to collect the bacterialcells.

The cells were transferred into the same jar fermenter as abovecontaining 2 liters of a medium containing 0.2% of N-AM, 0.1% of meatextract, 0.5% of polypeptone, 0.2% of yeast extract, 0.14% of sodiumchloride and 0.1% of monopotassium hydrogen phosphate and having a pHvalue of 6.8, and culture was continued under the same conditions asabove. Six hours after, activity of N-AMDH reached the maximum.

To 1.7 kg of alive bacterial cell obtained, 10 liters of 0.02Mtris-hydrochloric acid buffer (pH 8.0, hereinafter this is referred toas "standard buffer") was added, and then Triton X-100 and EDTA wereadded so that their concentration came to 0.5% and 2 mM, respectively.The mixture was stirred overnight in a cold room to obtain a uniformsuspension. It was milled by means of Dynomill (manufactured by SHINMARUEnterprise Co., Sweden) at 3,000 rpm and centrifuged at 8,000 rpm for 20minutes to obtain 7.3 liters of a supernatant liquid.

Then, 1.4 kg of DEAE-cellulose in wetness was added to the supernatantliquid, and the mixture was adjusted to pH 8.0 and stirred for 30minutes to have the enzyme adsorbed on the DEAE-cellulose. Aftertransferring it to Buchner's funnel and filtering it, it was washed with4 liters of standard buffer and then with 5 liters of standard buffercontaining 0.3M of sodium chloride. The fractions eluted with the lastwashing were combined and concentrated to one liter by means of hollowfiber ultrafiltrater (manufactured by Asahi Kasei Kogyo K. K.).

Into the concentrate was added and dissolved 125 g of ammonium sulfate.After thoroughly stirring the mixture and allowing it to stand for 2hours, it was centrifuged at 9,000 rpm for 20 minutes to obtain 850 mlof supernatant liquid. After adding an additional 166 g of ammoniumsulfate and thoroughly dissolving it, the mixture was left standingovernight in a cold room.

Then it was centrifuged at 12,000 rpm for 20 minutes to collect theresulting precipitate, and the latter was dissolved into 850 ml ofstandard buffer. After dissolving 35 g of ammonium sulfate thereinto,the resulting solution was passed through a column (5 cm in diameter and34 cm in height) of Phenyl-Sepharose CL-4B (manufactured by PharmaciaFine Chemicals, Sweden) previously equilibrated with standard buffercontaining 4% of ammonium sulfate to have the enzyme adsorbed on thecolumn. The enzyme was eluted with 10 liters of standard buffer having aconcentration gradient of ethylene glycol (0 to 30%) and a reverseconcentration gradient of ammonium sulfate (4 to 0%) at the same time.

The eluate was concentrated by ultrafiltration and dialyzed againststandard buffer containing 0.1M sodium chloride. Then it was passedthrough a DEAE-Sephadex A-50 column (5 cm in diameter and 52 cm inheight) previously equilibrated with standard buffer containing 0.1Msodium chloride for the sake of adsorption, and then the adsorbed matterwas eluted with 10 liters of standard buffer having a sodium chlorideconcentration gradient ranging from 0.1M to 0.28M.

The active fraction was concentrated by ultrafiltration and thendialyzed against 0.01M potassium phosphate buffer (pH 6.5). Then it waspassed through a column (4 cm in diameter and 16 cm in height) of5'-AMP-Sepharose CL-4B (manufactured by Pharmacia Fine Chemicals,Sweden) previously equilibrated with 0.01M potassium phosphate buffer(pH 6.0) for the sake of adsorption, and the adsorbed matter was elutedwith a buffer having sodium chloride concentration gradient (0-0.5M) andpH gradient (6.0-8.0) at the same time, by the use of 4 liters of 0.01Mphosphate buffer (pH 6.0) and 4 liters of 0.01M phosphate buffercontaining 0.5M sodium chloride (pH 8.0).

The active fraction was adjusted to pH 8.0 and then concentrated to 1 mlby means of ultra filter and collodion bag concentration apparatus, andthe concentrate was dialyzed against standard buffer containing 0.1Msodium chloride. The dialyzed solution was subjected to gel filtrationthrough a Sephadex G-200 column (2.5 cm in diameter and 95 cm in height)previously equilibrated with standard buffer containing 0.1M sodiumchloride.

The active fractions were combined and concentrated to obtain 2200 unitsof purified N-AMDH. As shown in FIG. 5, it was an enzyme sampleexhibiting a nearly single band in disk electrophoresis.

EXAMPLE 2

In the same manner as in Example 1, Flavobacterium sp. No. 141-8 wastransplanted into 50 ml of a seed culture medium (pH 6.8) containing0.5% of N-AM, 0.8% of polypeptone, 0.1% of meat extract, 0.2% of yeastextract and monopotassium hydrogen phosphate and subjected to a shakingculture at 30° C. for 24 hours.

The culture fluid was transplanted into a miniature jar fermentercontaining the same medium as above, and subjected to aeration (2liters/minute) agitation (400 rpm) culture at 30° C. for 40 hours.N-AMDH was accumulated in the bacterial cells. The bacterial cells thusobtained were treated in the same manner as in Example 1 to obtain anN-AMDH sample.

EXAMPLE 3

Concentration of N-AM in the solution was determined with the followingreagents.

    ______________________________________                                        1.   Reagents                                                                 0.1M phosphate buffer (pH 8.0)                                                                        880    microliters                                    NAD (60 mM)             50     microliters                                    N-AMDH (123 units/ml)   30     microliters                                    Sample solution         20     microliters                                    2.   Method of determination                                                  ______________________________________                                    

Predetermined quantities of the reagents were taken into a test tube andreacted at 37° C. for 10 minutes, after which absorbance was measured at340 nm. In a blank test, the above-mentioned procedure was repeated,except that the sample solution was replaced with an identical quantityof water. Absorbance of the blank run was subtracted from that of thesample run, and the remainder was taken as absorbance of samplesolution. Apart from above, N-AM solutions having known concentrationswere treated in the same manner as above, from which a calibration curvewas prepared. From the calibration curve, a concentration of N-AM inunknown sample was determined. FIG. 6 is the calibration curve.

EXAMPLE 4

Concentration of N-AM in solution was determined by the following methodby the use of the following reagents.

    ______________________________________                                        1.  Reagents                                                                  0.1M Phosphate buffer (pH 8.0)                                                                        140    microliters                                    (containing 0.1% Triton X-100)                                                Phenazine methosulfate (1 mg/ml)                                                                      5      microliters                                    Nitroblue tetrazolium (10 mg/ml)                                                                      5      microliters                                    NAD (40 mg/ml)          20     microliters                                    N-AMDH (123 units/ml)   10     microliters                                    Sample solution         10     microliters                                    2.   Method of determination                                                  ______________________________________                                    

Predetermined quantities of the above-mentioned reagents were taken intoa test tube and reacted at 37° C. for 15 minutes. Then, 2.0 ml of 0.3Nhydrochloric acid was added and thoroughly stirred. Absorbance of theformed color was measured at 570 nm. In a blank test, the reaction wasrepeated, except that the sample solution was replaced with identicalquantity of water. Absorbance of blank solution was subtracted from thatof the sample solution, and the remainder was taken as absorbance ofsample. Apart from above, a calibration curve was prepared fromsolutions having known N-AM concentrations. From the calibration curve,N-AM concentration in sample solution was determined.

EXAMPLE 5

Quantity of SA in serum was determined by the following procedure withthe following reagents.

    ______________________________________                                        1.    Reagents                                                                A.      10mM Phosphate buffer (pH 6.6)                                                                       1     ml                                               Neuraminidase (5 units/ml)                                                                           1     ml                                               N-Acetylneuraminic acid aldolase                                                                     1     ml                                               (10 units/ml)                                                         B.      0.1M Phosphate buffer (pH 8.0)                                                                       5.7   ml                                               N-AMDH (123 units/ml)  0.6   ml                                               NAD (60 mM)            0.5   ml                                               Oxamic acid            4.4   mg                                       2.    Method of determination                                                 ______________________________________                                    

Twenty microliters of serum was taken into a test tube, to which wasadded 300 microliters of reagent A. After reacting them at 37° C. for 15minutes, 680 microliters of reagent B was added and reacted for anadditional 10 minutes. Absorbance of the reaction mixture was measuredat 340 nm. In blank test, the above-mentioned procedure was repeatedexcept that reagent A was replaced with water. Absorbance of blank runwas subtracted from that of the sample run. A calibration curve wasprepared from solutions containing known concentrations ofN-acetylneuraminyllactose. From the calibration curve, SA concentrationin sample was determined.

EXAMPLE 6

Activity of N-acylglucosamine-2-epimerase extracted from hog kidney wasdetermined by the following procedure with the following reagents.

    ______________________________________                                        1.  Reagents                                                                  A.    0.5M Tris-hydrochloric acid buffer                                                                  25     microliters                                      (pH 7.4)                                                                      0.1M MgCl.sub.2       25     microliters                                      0.1M Acetylglucosamine                                                                              100    microliters                                      0.1M ATP (adjusted to pH 7.6)                                                                       10     microliters                                B.    0.1M Tris-hydrochloric acid buffer                                                                  700    microliters                                      (pH 8.2)                                                                      N-AMDH (123 units/ml) 30     microliters                                      NAD (60 mM)           100    microliters                                2.  Method of Determination                                                   ______________________________________                                    

An enzyme solution extracted from hog kidney (Biochemistry, 9, 3363) wasdissolved into 20 mM potassium phosphate buffer (pH 7.6), and 10microliters of the resulting solution was reacted with reagent A at 37°C. for 20 minutes. It was heated at 100° C. for 2 minutes and thencooled to 37° C., after which a predetermined amount of reagent B wasadded and reacted for 5 minutes. Absorbance of the reaction mixture wasmeasured at 340 nm. In blank test, the above-mentioned procedure wasrepeated, except that the enzyme solution was replaced with water.Absorbance of the blank run was subtracted as control from that ofsample run, and the remainder was taken as the increase of absorbanceattributable to the formed N-AM. Based on the fact that 1 mM of N-AMcorresponded to an absorbance of 6.27 in the reaction mixture,concentration of N-AM was calculated, and it was taken as the amount ofN-AM formed during 20 minutes. By converting it into "formation per onehour" and multiplying the latter by 100, enzyme activity per 1 ml ofenzyme solution was determined.

EXAMPLE 7

Concentration of SA in a solution was determined by the followingprocedure with the following reagents.

    ______________________________________                                        1.  Reagents                                                                  0.1M Phosphate buffer (pH 8.0)                                                                        610    microliters                                    N-Acetylneuraminic acid aldolase                                              (manufactured by Nakarai Kagaku                                               K. K.) (10 units/ml)    300    microliters                                    NAD (60 mM)             53     microliters                                    N-AMDH (123 units/ml)   60     microliters                                    Sample solution         20     microliters                                    2.  Method of determination                                                   ______________________________________                                    

Predetermined quantities of the reagents were taken into a test tube andreacted at 37° C. for 10 minutes, after which absorbance of the reactionmixture was measured at 340 nm. In blank test, the above-mentionedprocedure was repeated, except that N-acetylneuraminic acid aldolase wasreplaced with identical quantity of water. Absorbance of the blank runwas subtracted from that of sample run, and the remainder was taken asan absorbance of sample solution. Apart from the above, a calibrationcurve was prepared by treating SA solutions of known concentrations inthe same manner as above. From the calibration curve, SA concentrationin sample solution was determined. FIG. 7 is the calibration curve.

EXAMPLE 8

Concentration of SA in solution was determined by the followingprocedure with the following reagents.

    ______________________________________                                        1.  Reagents                                                                  0.1M Phosphate buffer (pH 8.0)                                                                        100    microliters                                    (containing 0.1% Triton X-100)                                                Phenazine methosulfate (1 mg/ml)                                                                      5      microliters                                    Nitroblue-tetrazolium (10 mg/ml)                                                                      5      microliters                                    NAD (40 mg/ml)          20     microliters                                    N-Acetylneuraminic acid aldolase                                                                      40     microliters                                    (manufactured by Nakarai Kagaku                                               K.K.) (10 units/ml)                                                           N-AMDH (123 units/ml)   10     microliters                                    Sample solution         10     microliters                                    2.  Method of determination                                                   ______________________________________                                    

Predetermined quantities of the above-mentioned reagents were taken intoa test tube and reacted at 37° C. for 15 minutes. After adding 2.0 ml of0.3N hydrochloric acid, it was thoroughly stirred. Absorbance of theformed color was measured at 570 nm. In blank test, the above-mentionedtreatment was repeated, except that N-acetylneuraminic acid aldolase wasreplaced with identical quantity of water. Absorbance of the blank runwas subtracted as control from that of the sample run, and the remainderwas taken as an absorbance of sample. Apart from above, a calibrationcurve was prepared by treating SA solutions of known concentrations inthe same manner as above. From the calibration curve, SA concentrationin sample solution was determined.

WHAT IS CLAIMED IS:
 1. A method for quantitatively analyzingN-acetylmannosamine in a sample which comprises reactingN-acetylmannosamine dehydrogenase with said sample containingN-acetylmannosamine and measuring NADH formed.
 2. A quantitativeanalysis kit for N-acetylmannosamine comprising N-acetylmannosaminedehydrogenase, NAD and a buffer solution.
 3. A method according to claim1, wherein absorbance at 340 nm is measured.
 4. A method according toclaim 1, wherein said N-acetylmannosamine dehydrogenase is obtained byculturing bacterial strain Flavobacterium sp. No. 141-8 (FERM BP-1222).5. A method for quantitatively analyzing sialic acid in a sample whichcomprises reacting said sample containing sialic acid withN-acetylneuraminic acid aldolase and N-acetylmannosamine dehydrogenaseafter successively or simultaneously and measuring NADH formed.
 6. Amethod according to claim 5, wherein said sialic acid is brought into aliberated state by reacting said sample containing sialic acid withneuraminidase.
 7. A kit for quantitatively analyzing sialic acidcomprising N-acetylneuraminic acid aldolase, N-acetylmannosaminedehydrogenase, NAD and a buffer solution.